Summary The transcription factor RUNX1 plays essential roles in definitive hematopoietic stem cell (HSC) ontogeny, HSC maintenance, megakaryocyte (Mk) maturation, and lymphocyte differentiation. RUNX1 deficiency causes an imbalance of HSC and progenitor cells, and is an early initiating step in up to 30% of all human leukemias. RUNX1 is also a recurrent target of heterozygous inactivating mutations in high-risk myelodysplastic syndrome (MDS). Despite RUNX1's central role in normal and malignant human hematopoiesis, its regulatory mechanisms remain incompletely understood. This gap in knowledge has impeded efforts to exploit RUNX1 as a therapeutic target. The long-term goal of our research is to elucidate these mechanisms and translate them into new treatment strategies. Our prior work and that of others in the field indicates that RUNX1 assembles into large dynamic multiprotein complexes that modulate its activity. These interactions involve other transcription factors, epigenetic regulators, and signaling enzymes. We hypothesize that these interactions are modulated by cell signaling pathways and that pharmacologic manipulation of these pathways can be used to enhance residual RUNX1 activity in disorders associated with partial RUNX1 deficiency. This is based on our preliminary studies demonstrating steady-state inhibition of RUNX1 activity by src-family kinases (SFKs), and synergistic RUNX1:Ets transcription factor interactions involving a region targeted by MEK/ERK-mediated phosphorylation. The following aims have been developed to test our central hypothesis: (1) identify changes in RUNX1 multiprotein complex formation that occur during cellular maturation and correlate them with RUNX1 activity; (2) determine how SFK and ERK signaling pathways modulate RUNX1 interactions with chromatin remodeling complexes/transcription factors and how they affect RUNX1 target gene expression; (3) Explore whether pharmacologic enhancement of residual RUNX1 activity can alleviate the HSC/progenitor cell imbalance observed with partial RUNX1 deficiency and impact leukemogenesis in mouse models of RUNX1 dominant negative fusion molecules. The results of these studies should fill in important gaps in knowledge regarding normal RUNX1 regulatory mechanisms and enable exploitation of RUNX1 as a therapeutic target in hematologic disorders. This work has the potential for immediate impact, as small molecule inhibitors of signaling pathways we hypothesize to impact RUNX1 activity are already clinically available and/or in testing.